The first step in the invasion of a pathogenic bacterium is attachment to its host. Attachment is often mediated by hairlike appendages that radiate out from the bacterial surface, called pili. Understanding pilus assembly will provide an important avenue of defense against bacterial- related diseases, and will also provide insight into the fundamental biological process of organelle biogenesis. This proposal aims to reconstitute pilus biogenesis in a cell-free system using defined components. P pili from uropathogenic Escherichia coli, a well studied model pilus, will be used for reconstitution. P pili are complex structures composed of at least six different subunits, and require two specialized assembly proteins: an outer membrane usher and a periplasmic chaperone. The outer membrane usher will be incorporated into liposomes, and pilus subunits will then be trapped inside or added to the outside of these proteoliposomes. Binding of chaperone-subunit complexes to the usher protein, assembly of pilus substructures, and assembly of complete pili will be examined. Successful reconstitution of pilus biogenesis will provide a powerful tool for analyzing mutants and determining the function of the subunits and assembly proteins. Reconstitution will facilitate the understanding of pilus-host cell interactions, and will aid in the design and testing of antimicrobial agents.